Structural Basis of Sterol Binding by NPC2, a Lysosomal Protein Deficient in Niemann-Pick Type C2 Disease

Abstract

NPC2 is a small lysosomal glycoprotein that binds cholesterol with submicromolar affinity. Deficiency in NPC2 is the cause of Niemann-Pick type C2 disease, a fatal neurovisceral disorder characterized by accumulation of cholesterol in lysosomes. Here we report the crystal structure of bovine NPC2 bound to cholesterol-3-O-sulfate, an analog that binds with greater apparent affinity than cholesterol. Structures of both apo-bound and sterolbound NPC2 were observed within the same crystal lattice, with an asymmetric unit containing one molecule of apoNPC2 and two molecules of sterol-bound NPC2. As predicted from a previously determined structure of apoNPC2, the sterol binds in a deep hydrophobic pocket sandwiched between the two ␤-sheets of NPC2, with only the sulfate substituent of the ligand exposed to solvent. In the two available structures of apoNPC2, the incipient ligand-binding pocket, which ranges from a loosely packed hydrophobic core to a small tunnel, is too small to accommodate cholesterol. In the presence of sterol, the pocket expands, facilitated by a slight separation of the ␤-strands and substantial reorientation of some side chains, resulting in a perfect molding of the pocket around the hydrocarbon portion of cholesterol. A notable feature is the repositioning of two aromatic residues at the tunnel entrance that are essential for NPC2 function. The NPC2 structures provide evidence of a malleable binding site, consistent with the previously documented broad range of sterol ligand specificity.

NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes

“…4A Inset and data not shown). The human P120S mutation replaces a residue that lies at the edge of the hydrophobic cholesterol-binding pocket as indicated by the crystal structure of the bovine NPC2 (11). Indeed, as shown in Fig.…”

“…NPC2 has been shown to bind cholesterol and derivatives such as cholesterol sulfate (8)(9)(10). X-ray crystallography reveals that cholesterol sulfate binds in such a manner that the iso-octyl side chain of the sterol is deeply buried in a hydrophobic pocket and the sulfate on the 3 position of the A ring is exposed to solvent (11). Introduction of a hydrophilic moiety, such as a hydroxyl group, on the cholesterol side chain prevents binding (10).…”

NPC1/NPC2 function as a tag team duo to mobilize cholesterol

“…Careful molecular dissection of NPC1 revealed that the soluble NTD of NPC1 [NPC1(NTD)] contained all of the sterol binding capacity of NPC1 and bound oxysterol stronger than cholesterol (9). This finding was in contrast to the soluble NPC2 protein that clearly showed a strong preference for cholesterol and did not bind oxysterols (8)(9)(10). Mutation of either NPC1 or NPC2 yields an identical Niemann-Pick disease etiology (1,5,6,12), suggesting that they work together in an unknown fashion to export cholesterol.…”

“…Whether NPC1 functions as a transporter remains controversial. In contrast, NPC2 is a soluble, glycosylated protein that is present in the lumen of the LE (5,8) (Fig. 1).…”

Clues to NPC1-mediated cholesterol export from lysosomes

“…Mutant NPC1 cells show massive accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes. NPC2 is a cholesterol binding protein that binds cholesterol via its iso-octyl moiety (9) (Fig. 1).…”